Project Summary Nanog Structure and Function in Stem Cell Pluripotency This proposed research will determine the structural basis for interactions made by Nanog, an epigenetic reprogramming factor that regulates cellular proliferation and differentiation. Nanog is required for self-renewal of embryonic stem cells and for re-programming, the transformation of somatic cells to pluripotent stem cells. Nanog interacts with >100 other proteins, but intrinsically disordered regions make Nanog difficult to study. The investigators will use their extensive experience with intrinsically disordered proteins to dissect Nanog function in two stages: they will determine how phosphorylation and self-assembly regulate Nanog stability and activity (Aim 1), and they will determine how interactions between Nanog and partners give rise to pluripotency (Aim 2). These experiments will establish the molecular mechanisms of Nanog hub function, and perhaps produce lead compounds for future drug discovery efforts to target Nanog-dependent cell proliferation and pluripotency, which is relevant to therapeutic interventions that utilize stem cells or that seek to destroy cancer stem cells. The first Aim will use solution NMR spectroscopy and other methods to reveal how phosphorylation in the Nanog PEST degradation signal alters local backbone conformation at Ser-Pro dipeptides and affects Nanog levels in cells via the phosphorylation-dependent proline isomerase Pin1. Reprogramming assays will be used to test the effects of perturbing Nanog/Pin1 interaction. Single molecule fluorescence methods will be used to characterize the structure and dynamics of the isolated Nanog C-terminal domain and to determine how the N- terminal domain modulates Nanog oligomerization in vitro and in vivo. These experiments will provide funda- mental insight into Nanog availability in the assembled state thought to underlie its function as a molecular hub. The second Aim will determine the mechanisms by which Nanog acts as the ?gateway to pluripotency?. The investigators will use unnatural amino acid photocrosslinking, chemical crosslinking and mass spectrometry- based proteomics to identify Nanog binding partners in stem cells, to uncover any correlations between multiple binding partners, and to map the Nanog sequences that support binding. They will determine the functional importance of these interactions by perturbing the partner and/or its binding site on Nanog in somatic cell reprogramming assays. These experiments will demonstrate the importance of key partners to Nanog function and will reveal which regions of Nanog recruit these partners to drive pluripotency. Both the partners and the regions of Nanog that they bind may be useful targets for modulating Nanog activity. They will determine how Nanog influences binding of the transcription factors Oct4 and Sox2 to each other and to the Nanog promoter. These experiments will reveal how Nanog scaffolds macromolecular assemblies that control stem cell pluripotency.